Abstract. Aquifers harbor unique and highly adapted species, contributing to critical ecological processes and services. Understanding the key factors driving invertebrate assemblages in aquifers is a challenging task that, traditionally, has primarily been achieved in karst aquifers. This study aimed to uncover the factors influencing the composition and functionality of groundwater crustaceans in a volcanic aquifer of central Italy. We adopted a multidisciplinary approach, combining hydrogeology, geology, microbiology, and ecology, and found that the aquifer consisted of three adjacent groundwater bodies (GWBs) with different geochemistry (i.e., sulfate-depleted, K-rich and earth-alkaline) and microbial characteristics that remained consistent over the study period. We also unveiled significant differences in both the taxonomic and functional composition of groundwater crustaceans across the three GWBs and these patterns were consistent over time. Notably, the sulfate-depleted GWB lacked groundwater-obligate species, burrowers, stenothermal and moderately stenothermal species, while the K-rich and earth-alkaline GWBs had different species with similar functions related to locomotion, diet, and feeding habits. Stenothermal and moderately stenothermal crustacean species were only found in the K-rich GWB, which lacked epigean species. Major ions (SO4, Ca, NO3, and K), trace elements (B, Al, V, Se, and Ba), and microbial factors related to microbial cells with low nucleic acid (LNA cells) and carbohydrate catabolic profiles were the main descriptors of groundwater-obligate species abundances. Our findings revealed a significant correlation between the abundances of groundwater-obligate crustaceans and LNA cells, thus suggesting a selective feeding of groundwater invertebrate species on the aquatic microbial community. Our research emphasizes the need to consider diverse hydrogeological contexts within individual aquifers. Potential avenues for future research should further consider food web dynamics in groundwater communities and their impact on carbon and nutrient cycling.